Treatment of cancers

ABSTRACT

A new treatment schedule for administration of N- 2-(dimethylamino)ethyl!acridine-4-carboxamide and other related carboxamide anticancer drugs in which the drug is administered in a divided-dose schedule comprising two or more administrations at frequent intervals, for example every hour. Schedules to produce cyclic peaks/troughs in plasma levels are mentioned. The compounds can be used for circumventing multidrug resistance in cancers and may, for example, be used in combination with other cytotoxic drugs, especially non-topo II inhibitors. Treatment of melanoma and advanced colon cancer is included.

This is a Rule 60 continuation of application Ser. No. 08/007,690, filedJan. 22, 1993, now abandoned.

The present invention relates to the treatment of tumours, especially tothe treatment of melanoma and cancer of the colon, and to thecircumvention of multidrug resistance in cancer treatments.

BACKGROUND OF THE INVENTION

For certain types of cancer, chemotherapy has been capable of renderingpatients with responsive tumours free of disease. However, thisresponsive category does not include the most frequently encounteredforms of malignant tumours.

The most common types of cancer in western populations are colon, lungand breast cancer. Each of these can be treated to some extent withexisting chemotherapy, with different drugs being used preferentiallyfor each type of malignancy (for instance, doxorubicin, cyclophosphamideand methotrexate for breast cancer, 5-fluorouracil for colon cancer),but response rates are not good. In addition, melanoma is a diseasewhich is increasing in incidence at an alarming rate among fair-skinnedpopulations. In melanoma, only 25-30% of patients with disseminateddisease respond to treatment, and only 5-10% sustain durable remission(Evans B. D., et al., Proc. Am. Soc. Clin. Oncol. 1990, 9, 276).

There is therefore a great need for new types of cancer therapy, and adesperate need for such treatments for the above cancers in particular.

The basis for the development of the majority of anticancer drugs usedtoday has been a panel of mouse tumours including transplantableleukaemias, the Lewis lung carcinoma and the colon 38 adenocarcinoma. Anumber of human tumour xenografts in mice have also been used. Ingeneral, the leukaemias are the most sensitive to experimental agents,the xenografts are the most resistant and the Lewis lung and colon 38are of intermediate sensitivity (Goldin A., et al., Eur. J. Cancer 1981,17, 129-142).

The murine Lewis lung adenocarcinoma is a tumour which initially arosespontaneously in C₅₇ Bl mice and which has a number of features whichmake it a good model for clinical carcinomas. It grows easily both invitro and in vivo, and is aneuploid, heterogeneous, metastatic andresistant to many but not all clinical antitumour agents. Zacharski(Zacharski L. R., Haemostasis 1986, 16, 300-320) has concluded thatalthough Lewis lung has the cytological appearance of a large-cellcancer, its rapid rate of growth, propensity to cause lethal metastases,as well as its susceptibility to combination chemotherapy, radiation andanticoagulant treatment, make it a good model for human small-cell lungcancer (SCLC). The colon 38 tumour arose in carcinogen-treated mice, andbecause it is sensitive to 5-fluorouracil it can be considered as auseful model for human colon cancer (Corbett T. H., et al., CancerChemother. Rep. 1975, 5, 169-186, and Cancer 1977, 40, 2660-2680). Humanmelanoma xenografts have been considered for some time as an appropriatemodel for the development of new anticancer drugs for melanoma (TaetleR., et al., Cancer 1987, 60, 1836-1841).

The essence of treating cancer with cytotoxic anticancer drugs is tocombine a mechanism of cytotoxicity with a mechanism of selectivity fortumour cells over host cells. The selectivity of a drug for a particularcancer will depend on the expression by that cancer of properties whichpromote drug action, and which differ from tumour to tumour.

Currently available cytotoxic drugs can be broadly divided into fourgroups: those which react chemically with DNA (such as the alkylatingagents and cisplatinum), those which disrupt DNA synthesis (such as theanti-metabolites), those which disrupt the mitotic apparatus (such asthe Vinca alkaloids) and those which are directed against the cellularenzyme topoisomerase II ("topo II") in order to effect changes in thetopological form of the DNA.

DNA topoisomerases were named after the first method used to detecttheir activity. When incubated with closed circles of double-strandedDNA prepared from viruses or bacteria, topoisomerases enzymaticallychange the number of coils contained in each circle (circular forms ofDNA with different degrees of coiling are called topo-isomers). Thetopoisomerases are perhaps better understood as enzymes whichtemporarily break one strand of the DNA double helix (topoisomerase I or"topo I") or which simultaneously break two strands of the DNA doublehelix ("topo II") in order to effect changes in the topological form ofthe DNA.

Topoisomerases have two main functions in the cell. The first is to actas swivel points on the DNA in association with DNA and RNA polymerasesduring the biosynthesis of nucleic acids required for cell replicationand gene expression. The second is to untangle the DNA strands of thedaughter chromosomes following DNA replication prior to cell division.The DNA of chromosomes is organised as a series of loops on aprotein-aceous "scaffold". After duplication of chromosomes and of the"scaffolds", the DNA loops must be separated. Since there are hundredsof thousands of DNA loops attached to each chromosome scaffold it is nothard to imagine the necessity for an enzyme which effectively removestangles by passing one double DNA strand through another. This processabsolutely requires topo II.

Topo I acts by transiently breaking a DNA strand and attaching itself toone of the free ends of the broken DNA via the amino acid tyrosine. TopoII contains two identical protein subunits, each of which is capable ofbreaking a DNA strand and attaching itself to one of the free ends. Withboth DNA strands broken, a second DNA double helix can be allowed topass between the two enzyme protein subunits, thus allowing not onlyswivelling but also untangling of DNA. The process is normallyspontaneously reversible by cleavage of the enzyme-DNA links andre-sealing of DNA breaks to restore the DNA to its original form.

Topo II-directed agents include a number of important clinicalanti-cancer drugs such as anthracycline antibiotics (e.g. doxorubicin),epipodophyllotoxin derivatives (e.g. etoposide) and synthetic DNAintercalating drugs (e.g. amsacrine). These act by jamming the enzyme inits DNA-associated form (Liu L. F., Annu. Rev. Biochem. 1989, 58,351-375). Such molecular lesions might be expected to be innocuous,since the drug eventually dissociates itself from the complex and theDNA strand breaks are then repaired perfectly. However, in a smallproportion of cases, the presence of drug causes the complex to bedissociated abnormally, generating some kind of DNA lesion whicheventually leads to cell death.

Although the antitumour activity of many of these agents has been knownfor many years, it is only since 1984 that the molecular target ofaction has been identified (Nelson E. M., Tewey K. M., Liu L. F., Proc.Natl. Acad. Sci. USA 1984, 81, 1361-1364 and Tewey K. M., et al.,Science 1984, 226, 466-468).

A number of mechanisms of resistance to topo II poisons have now beenidentified, and in many cases the development of resistance to one drugis accompanied by the simultaneous acquisition of resistance to avariety of other drugs. Since the mechanism of resistance determines thepattern of cross-resistance to other drugs, an understanding of theseprocesses is of great importance to the strategy for the use of theseagents. Several resistance mechanisms important to the use of theseagents have now been characterised in experimental systems, includingthose involved in drug transport (Endicott J. A., Ling V., Annu. Rev.Biochem 1989, 58, 351-375), drug-target interaction (Beck W. T.,Biochem, Pharmacol. 1987, 36, 2879-2888) and drug detoxification (DeffieA. M., et al., Cancer Res. 1988, 48, 3595-3602).

Attempts to overcome multidrug resistance (mdr) clinically have beenconcerned mainly with the first of these mechanisms, a drug transportmechanism that pumps drug out of cells. Various inhibitors of thisprocess, such as verapamil, are known and some have been used incombination with drugs such as doxorubicin and etoposide to treat cancer(Stewart D. J., Evans W. K., Cancer Treat. Rev. 1989, 16, 1-10, JudsonI. R., Eur. J. Cancer 1992, 28, 285-289).

Another approach is to design drugs which can overcome mdr. We have nowdiscovered that the investigational drug acridine carboxamide ("DACA")appears to be one such drug.

The compound tested was the dihydrochloride of N-2-(dimethylamino)ethyl!acridine-4-carboxamide of formula ##STR1## and isdescribed and claimed in EP 98098. That patent also describes and claimsother acridine carboxamide compounds and their use for the treatment oftumours; more particularly, the treatment of Lewis lung tumours andleukaemia is described.

Various other derivatives of DACA have been tested for their antitumouractivity and the results are reported in the literature. Active,compounds are carboxamides having an unsubstituted or substitutedaromatic ring system comprising two or more fused rings and having anoxygen or an aromatic nitrogen peri to the carboxamide side chain. Aswell as the other acridine carboxamides (EP 98098 and Denny W. A., etal., J. Med. Chem. 1987; 30: 658-663), examples are phenyl quinoline andpyrido quinoline carboxamides (EP 206802 A, Atwell G. J., et al., J.Med. Chem. 1988; 31: 1048-1052 and Atwell G. J., et al., J. Med. Chem.1989; 32: 396-401), phenazine carboxamides (EP 172744 A and Rewcastle G.W., et al., J. Med. Chem. 1987; 30: 843-851), carboxamides havingangular tricyclic chromophores: phenanthridine carboxamides (NZ Patent215286, 1986 and Atwell G. J., et al., J. Med. Chem. 1988; 31: 774-779)and carboxamides having various linear tricyclic chromophores (Palmer B.D., et al., J. Med. Chem. 1988; 31: 707-712). These other compounds arestructurally very similar to DACA and are able to act in the same way.

DACA is a DNA-binding drug which acts at the same target, topoisomeraseII, as do drugs such as amsacrine and etoposide. We have now found thatit has a different in vitro cytotoxicity profile to these compounds anda number of advantages over existing clinical drugs in the class oftopoisomerase-directed agents.

Firstly, it is active against cell lines displaying bothP-glycoprotein-mediated or "transport" resistance and "atypical" or"altered" multidrug resistance; in this respect it is unique among topoII inhibitors.

DACA and related compounds may therefore be used to circumvent mdr. Forthis it may be used in combination with other cytotoxic drugs, moreespecially non-topo II inhibitors, and/or as a second-line treatment iffirst-line treatment fails because of the development of multidrugresistance.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further explained with reference to the attacheddrawing in which:

FIG. 1 is a graph showing comparisons of DELTA values in log mean graphformat for DACA, amsacrine, etoposide, doxorubicin, vincristine,5-fluorouracil, camptothecin and mitozantrone;

FIG. 2 is a scattergram showing the transport multidrug resistanceagainst the susceptibility to topoisomerase multidrug resistance for thesame 6 drugs of FIG. 1;

FIG. 3 is a plot of DELTA values of DACA compared with amsacrine,etoposide and doxorubin using protein staining;

FIG. 4 is a plot of the DELTA values in a series of 12 primary melanomacultures comparing DACA with the three other topo II agents as in FIG.3;

FIG. 5 is a graph showing a single maximum tolerated dose of DACA versusfour injections at 30 minute intervals comparing relative tumor volumeover time;

FIG. 6 is a graph showing DACA administered in divided doses at 0 and 7days against controls comparing relative tumor volume versus treatmenttime in days; and

FIG. 7 is a graph showing toxicity versus cell-associated drug relatingcell kill per hour against drug concentration comparing the experimentaldata with a theoretical model.

DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides the use of DACA or otheraromatic fused-ring carboxamide having an aromatic nitrogen atom or anoxygen atom peri to the carboxamide side chain or a physiologicallytolerable acid addition salt thereof, for the manufacture of amedicament for overcoming mdr.

The present invention further provides a method of overcoming mdr,wherein there is administered DACA or other aromatic fused-ringcarboxamide having an aromatic nitrogen atom or an oxygen atom peri tothe carboxamide side chain or a physiologically tolerable acid additionsalt thereof.

The present invention further provides a pharmaceutical preparationcomprising

(i) DACA or other aromatic fused-ring carboxamide having an aromaticnitrogen atom or an oxygen atom peri to the carboxamide side chain or aphysiologically tolerable acid addition salt thereof,

and

(ii) a DNA-reactive agent, a DNA-synthesis inhibitor or an agent whichdisrupts the mitotic apparatus, in admixture or conjunction with apharmaceutically suitable carrier.

The present invention also provides a combined preparation for use inthe treatment of cancer, comprising separate components (i) and (ii)above for simultaneous or sequential administration.

Secondly, we have found that, unexpectedly, DACA is effective againstadvanced colon 38 tumours and an advanced melanoma xenograft in micecolon when administered in a divided dose schedule over a period of twohours. (In this context "advanced tumour" means that the tumour was morethan 5 mm in diameter at the time of measurement.) In contrast, a singleadministration of DACA at the maximum tolerated dose (150 mg/kg), whichis curative against Lewis lung tumours growing as lung nodules in mice,was only marginally effective.

Thirdly, DACA has the ability to cross the blood brain barrier,suggesting that rapidly growing brain tumours may also be treatable,more especially when administered in a divided dose schedule.

Accordingly, the present invention provides the use of DACA or otheraromatic fused-ring carboxamide having an aromatic nitrogen atom or anoxygen atom peri to the carboxamide side chain or a physiologicallytolerable acid addition salt thereof, for the manufacture of amedicament for the treatment of detectable colon cancer, melanoma orbrain tumours, more especially by administration of a divided dose, theconstituent doses being administered at frequent intervals.

The present invention further provides a method for the treatment ofdetectable colon cancer, melanoma or brain tumours, wherein there isadministered DACA or other aromatic fused-ring carboxamide having anaromatic nitrogen atom or an oxygen atom peri to the carboxamide sidechain or a physiologically tolerable acid addition salt thereof, moreespecially by administration of a divided dose, the oenstituent dosesbeing administered at frequent intervals.

There may, for example, be at least 2 administrations in total in thedivided dose, administrations being at least every 2 hours, for exampleevery hour or every 1/2 hour, for up to 4 hours.

Thus, the present invention also provides the use of DACA or otheraromatic fused-ring carboxamide having an aromatic nitrogen atom or anoxygen atom peri to the carboxamide side chain or a physiologicallytolerable acid addition salt thereof, for the manufacture of adivided-dose medicament for the treatment of tumours, including melanomaand colon and brain tumours, by a treatment regime comprising 2 to 4administrations of drug over a period of up to 4 hours, for example 2 to4 hours.

The present invention also provides a method for the treatment oftumours, including melanoma and colon tumours, which comprises theadministration of a divided dose of DACA or other aromatic fused-ringcarboxamide having an aromatic nitrogen atom or an oxygen atom peri tothe carboxamide side chain or a physiologically tolerable acid additionsalt thereof, 2 to 4 constituent doses being administered over a periodof up to 4 hours, for example 2 to 4 hours.

Fourthly, we believe that suitable DNA-binding compounds will reduce thetoxicity of DACA when administered in conjunction with a dividedhigh-dose schedule of DACA. DNA-binding compounds include, for example9-aminoacridine; such compounds have the ability to inhibit theantitumour activity of DACA.

Accordingly, the present invention provides the use of a DNA-bindingagent in combination with DACA or other aromatic fused-ring carboxamidehaving an aromatic nitrogen atom or an oxygen atom peri to thecarboxamide side chain or a physiologically tolerable acid addition saltthereof, to reduce the host toxicity of DACA or specified othercompound.

Doses in mice of 100 to 300 mg/kg, especially 150 to 250 mg/kg, moreespecially substantially 200 mg/kg, administered as a divided dose overa period of 2 to 4 hours, have proved suitable. Administration to humansof, for example, substantially 800 mg/m² of DACA or equivalent amount ofother carboxamide, should be mentioned, but lower or higher amounts mayalso be possible. As explained above, advantageous results are obtainedwhen the dose is administered as a divided dose, producing a high plasmalevel followed by a drop in level and then a high level again. Thus, theuse of substantially 800 mg/m² for the total of the constituent doses ofa divided dose should be mentioned.

Compounds suitable for use according to the present invention are thoseof the general formula

    ArCONH(CH.sub.2).sub.n Y                                   (I)

in which

Ar represents an unsubstituted or substituted ring system comprising twoor more fused aromatic rings and having an aromatic nitrogen atom or anoxygen atom peri to the carboxamide side chain,

Y represents C(NH)NH₂, NHC(NH)NH₂ or NR₄ R₅, where each of R₄ and R₅separately is H or lower alkyl optionally substituted by one or more ofthe same or different substituents selected from hydroxy, lower alkoxyand amino functions, or R₄ and R₅ together with the nitrogen atom towhich they are attached form a 5- or 6-membered heterocyclic ringoptionally containing a further hetero atom; and

n represents an integer from 2 to 6, and their physiologically tolerableacid addition salts and N-oxides thereof.

The ring system may comprise, for example, three fused aromatic rings,preferably linear, or two fused aromatic rings with a carbocyclic orheterocyclic aromatic ring as substituent. Of the fused aromatic rings,one or more may be heterocyclic.

In a preferred embodiment of the present invention there is used acompound of the general formula ##STR2## in which R₁ represents H, CH₃or NHR_(o), where R_(o) is H, COCH₃, SO₂ CH₃, COPh, SO₂ Ph or loweralkyl optionally substituted with hydroxy, lower alkoxy and/or aminofunctions;

R₂ represents H or lower alkyl, halogen, CF₃, CN, SO₂ CH₃, NO₂, OH, NH₂,NHSO₂ R₃, NHCOR₃, NHCOOR₃, OR₃, SR₃, NHR₃ or NR₃ R₃ (where R₃ is loweralkyl optionally substituted with hydroxy, lower alkoxy and/or aminofunctions), and/or may represent the substitution of an aza (--N═) groupfor one of the methine (--CH═) groups in the carbocyclic ring,

Y represents C(NH)NH₂, NHC(NH)NH₂ or NR₄ R₅, where each of R₄ and R₅separately is H or lower alkyl optionally substituted with hydroxy,lower alkoxy and/or amino functions, or R₄ and R₅ together with thenitrogen atom to which they are attached form a 5- or 6-memberedheterocyclic ring optionally containing a further hetero atom;

n represents an integer from 2 to 6;

X₁ represents H, and

X₂ represents a phenyl or pyridyl ring unsubstituted or substituted by asubstituent R₆, or

X₁ and X₂, together with the carbon atoms to which they are attached,form a fused benzene ring unsubstituted or substituted by a substituentR₆, and

R₆ represents lower alkyl, halogen, CF₃, CN, SO₂ CH₃, NO₂, OH, NH₂,NHSO₂ R₃, NHCOR₃, NHCOOR₃, OR₃, SR₃, NHR₃ or NR₃ R₃ (where R₃ is loweralkyl optionally substituted with hydroxy, lower alkoxy and/or aminofunctions); or a phenyl ring optionally further substituted by loweralkyl, halogen, CF₃, CN, SO₂ CH₃, NO₂, OH, NH₂, NHCOR₃, NHCOOR₃, OR₃,SR₃, NHR₃ or NR₃ R₃ (where R₃ is lower alkyl optionally substituted withhydroxy, lower alkoxy and/or amino functions); and/or may represent thesubstitution of an aza (--N═) group for one of the methine (--CH═)groups in the ring;

or a physiologically tolerable acid addition salt, or, especially whenX₁ ═H and X₂ is unsubstituted or substituted phenyl or pyridyl, a1-N-oxide thereof.

A compound of the general formula Ia in which X₁ and X₂ complete a fusedring and R₁ represents an unsubstituted or substituted phenyl groupshould also be mentioned.

Another class of compounds is, for example, represented by the generalformula Ib ##STR3## in which

R₇ represents H or up to three substituents, at positions selected from2 to 4 and 6 to 9, wherein any two or all of the substituents may be thesame or different and the substituents are selected from lower alkylradicals; lower alkyl radicals substituted by one or more of the same ordifferent substituents selected from hydroxy, lower alkoxy and/or aminofunctions; OH; SH; OCH₂ Ph; OPh; NO₂ ; halogen; CF₃ ; amino; NHSO₂ R₃,NHCOR₃, NHCOOR₃, OR₃ and SR₃ (where R₃ has the meaning given above; andCONH(CH₂)_(n') Y' (where n' and Y' are as defined below), there being amaximum of one CONH(CH₂)_(n') Y' group; or any two of R₇ at adjacentpositions represent --CH═CH--CH═CH-- as part of an extra benzene ring or--O--CH₂ --O-- (methylenedioxy) and the third of R₇ has any one of themeanings given above with the exception of an OH at position 2;

Y and Y', which may be the same or different, each has the meaning givenabove for Y; and n and n', which may be the same or different, each hasthe meaning given above for n;

and physiologically tolerable acid addition salts, 5- and 10-mono-N-oxides and 5,10-di-N-oxides thereof.

These compounds may be prepared by methods known per se, for example bymethods described in EP 98098 A, in EP 206802 A and in EP 172744 A or byanalogous methods.

When used herein, the term "lower alkyl" denotes an alkyl group havingfrom 1 to 5, preferably 1 to 4, carbon atoms.

An amino function as substituent of a lower alkyl radical represented byany of R₃, R₄, R₅, R_(o) and R₇ may be unsubstituted or, for example,substituted by one or two lower alkyl groups (where lower alkyl has themeaning given above), especially by one or two methyl groups. Thus, forexample, an amino substituent of a lower alkyl radical represented byR₃, R₄, R₅, R_(o) and/or R₇ may be NH₂, NHCH₃ or N(CH₃)₂.

A lower alkoxy group as substituent of a lower alkyl radical representedby R₃, R₄, R₅, R_(o) and/or R₇ is especially a methoxy group.

A heterocyclic radical represented by R₄ and R₅ and the nitrogen atomsto which they are attached may, if desired, contain an additional heteroatom, and is 5- or 6-membered. An example is a morpholino group.

Examples of optionally substituted lower alkyl groups include thosesubstituted by hydroxy, lower alkoxy or an amino function, for examplelower alkyl optionally substituted with hydroxy, amino, methylamino,dimethylamino or methoxy. when X₁ +X₂ complete a fused benzene ring suchlower alkyl groups are preferably unsubstituted or substituted withhydroxy and/or amino groups.

In a NR₃ R₃ group the two R₃ substituents may be the same or different,but are preferably the same.

A preferred class of compound of the above formula I where X₁ representsH and X₂ represents a phenyl or pyridyl ring is where R₁ represents H,and, more especially,

R₂ represents H,

Y represents N(CH₃)₂,

n represents 2 and

if X₂ represents a pyridyl ring, that ring is unsubstituted, and

if X₂ represents a phenyl ring that ring is unsubstituted or substitutedby halogen, NO₂ or OCH₃.

A pyridyl ring represented by X₂ is preferably a 4-pyridyl ring.

The use of an acridine carboxamide of the general formula ##STR4## whereR₁ and n have the meanings given above, R₈ represents H or up to two ofthe groups CH₃, OCH₃, halogen, CF₃, NO₂, NH₂, NHCOCH₃, and NHCOOCH₃placed at positions 1-3 and 5-8, and/or may represent the substitutionof an aza (--N═) group for one of the methine (--CH═) groups in thecarbocyclic ring;

and

Y represents C(NH)NH₂, NHC(NH)NH₂, or NR₄ R₅, where each of R₄ and R₅ isH or lower alkyl optionally substituted with hydroxyl and/or aminogroups;

and where any lower alkyl radical has up to 4 carbon atoms,

and the physiologically tolerable acid addition salts thereof, shouldespecially be mentioned.

A preferred subclass of these compounds of formula

I' are those where

R₁ represents H or NH₂,

R₈ represents up to two of 1-, 5-, 6-, 7- and 8-NO₂, 5- and 6-CH₃, and5-Cl,

Y represents NHC(NH)NH₂, N(CH₃)₂, or NHCH₂ CH₂ OH, and

n represents 2.

Compounds specifically identified in EP 98098 A, EP 206802 A and EP172744 A and in the literature references given above should also bementioned.

When any of R₂, R₆ and R₈ represents the substitution in the ring of anaza group for one of the methine groups, that ring may be unsubstitutedor substituted as specified above.

Compounds of the general formula Ia or I' in which R₆ or R₈ representsthe substitution of an aza group for one of the methine groups and whichoptionally contains a further R₆ or R₈ substituent(s) are novel, and assuch form part of the present invention.

The compounds used according to the invention, including compounds offormulae Ia and Ib, form pharmaceutically acceptable addition salts withboth organic and inorganic acids. Examples of suitable acids for saltformation are hydrochloric, sulphuric, phosphoric, acetic, citric,oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleicand methanesulphonic acids.

When used as a means of circumventing mdr, in combination with anothercytotoxic drug, for example a DNA-reactive agent, a DNA-synthesisinhibitor or an agent which disrupts the mitotic apparatus, the compoundof the general formula I may be administered together with, before orafter the other cytotoxic drug. DACA could be given, for example, up to±2 days of a second drug, or alternatively could be given as a separateor alternating course with another cytotoxic drug and separated by aperiod of bone marrow or other host tissue recovery, generally 3 to 4weeks. DACA may, for example, be administered by intravenous infusionusing the divided dose regime mentioned above, for example as a seriesof 2 to 4 administrations over a period of 2 to 4 hours.

Suitable DNA-reactive agents are, for example, cisplatin,cyclophosphamide, bleomycin and carboplatin. Suitable DNA synthesisinhibitors are, for example, 5-fluorouracil, 5-fluorodeoxyuridine andmethotrexate; and suitable agents that disrupt the mitotic apparatusare, for example, taxol and suitable Vinca alkaloids, for example,vincristine, vinblastine and vindesine. These agents should be used in atreatment schedule which has been found optimal for antitumour effect;for example, cyclophosphamide may be used at monthly intervals, andvincristine at monthly or weekly intervals.

There are four main types of multidrug resistance related to topo IIinhibitor:

(a) No change in the topoisomerase enzyme but increased transport of thedrug out of the cell. DACA is not susceptible to this, while etoposideand doxorubicin are.

(b) No change in the enzyme, but an increase in drug detoxifyingenzymes. This applies to doxorubicin but not to etoposide or DACA.

(c) A quantitative change (decrease) in the amount of topoisomerase IIenzyme. DACA, etoposide and doxorubicin are equally susceptible, sinceDNA damage depends on the amount of enzyme present. Since the amount oftopo II is regulated during the cell division cycle, cytokineticresistance, whereby non-cycling cells resist the effects of topo IIagents, may involve this type of resistance.

(d) A qualitative change in the topoisomerase enzyme, a result either ofa switch in gene expression (there are two genes for topoisomerase IIand one is normally dominant) or of a mutation in the gene, or of achange in modification of the enzyme after it has been synthesised. Thisresults in a change in drug-target interaction. The qualitative changeis accompanied by a differential change in sensitivity: in general,cells become highly resistant to amsacrine, moderately resistant toetoposide and doxorubicin, and, we have ascertained, minimally resistantto DACA.

Investigation of the cytotoxicity of DACA under conditions of continuousdrug exposure in a variety of human and mouse cell lines and in a panelof 60 human cell lines revealed IC₅₀ values (defined as drugconcentration required over approx. 5 cell doubling times for thereduction of the final cell culture density by 50%) ranging from 0.09 μMto 3.4 μM, and a mean IC₅₀ value for human cell lines of 1.3 μM. Thelatter value compared with 2 μM for the 4-pyridyl quinoline analogue,0.76 μM for amsacrine, 0.1 μM for the amsacrine analogue 4'-(9- 4-N-methylcarboxamido!-5-methyl!-acridinylamino)methanesulphon-m-anisidide("CI-921") and 81 μM for etoposide. Whereas the patterns of cytotoxicityof amsacrine, CI-921 and etoposide in the human cell line panel werevery similar, those of DACA and its pyridoquinoline analogue were quitedifferent, suggesting differences in mode of action.

A multidrug resistant subline of P388 murine leukaemia (P/ACTD) wastested for sensitivity to DACA. This line was cross-resistant toactinomycin D, doxorubicin, mitoxantrone, etoposide and vincristine. Itsresistance to vincristine was overcome by the presence of verapamil (10μM). It stained for the presence of P-glycoprotein, consistent with thepresence of transport-mediated multidrug resistance. This line wassensitive to DACA in vitro and in vivo, suggesting that DACA may beuseful in at least some types of multidrug resistance.

DACA was also able to overcome, to a large extent, other mechanisms ofmultidrug resistance, as demonstrated in a series of sublines of Jurkatleukaemia cells which were highly resistant to amsacrine, etoposide anddoxorubicin. Two of these lines had been selected for resistance toamsacrine, and were more than 100-fold cross-resistant to amsacrine butonly 2- to 4-fold cross-resistant to DACA. These lines exhibitedresistance mechanisms which were distinguishable from transport-mediatedmultidrug resistance. We believe that this ability to overcomeresistance mechanisms accounts for the different IC₅₀ patterns observedwith the human cell line panels.

It is apparent that in many tumours, regrowth during therapy isassociated with resistance. The type of resistance is not yet properlycharacterised, but if it involves the mechanisms discussed above, DACAmay be useful for second time treatment, especially in the divided doseregime mentioned above.

The use of the above compounds and combinations in the treatment ofsarcoma and of lung, breast, ovarian and testicular cancer shouldespecially be mentioned.

The use of compounds of the general formula I to treat colon tumours hasbeen suggested previously, but there has been no evidence of theirsuitability for this treatment and there has been no indication thatthey are effective in test systems even with delay of initiation oftreatment beyond day 2 or 3 after tumour implantation, as is usual intests. There has also been no disclosure of high activity against suchtumours. Such high activity would not have been expected since the mostclosely structurally related topo II inhibitor, amsacrine, is inactive.

An initial experiment against advanced colon 38 tumour (on day 11 afterimplantation), using the same schedule of administration as used forLewis lung (3 injections at 4-day intervals) gave only a modest growth.delay (4 days).

However, by adjustment of the administration schedule of DACA, growthdelay of the advanced colon 38 tumour (5-8 mm in diameter) was increasedto more than 21 days. Thus, while intermittent schedules (270 mg/kg q⁴days×3; 400 mg/kg q⁷ days×3) provided only modest growth delays (3 daysand 7 days, respectively), repeated injection schedules (4 injections at30 minute intervals; 180+120+120+120 μmol/kg, q⁷ days×3) provided a 21day growth delay. Such results were completely unexpected.

We have found that a low drug concentration for a long time (for example6 hours) is much more toxic than a high concentration for acorrespondingly shorter time. We believe these unusual "self-inhibitory"properties of DACA may be of help in the new divided dose administrationstrategy. Because DACA diffuses more slowly in solid tumours such ascolon tumours, than in normal tissues, peak drug concentrations intumours are lower than in normal tissues: an obvious disadvantage.However, because, as we have found, higher concentrations of DACA areless inhibitory than lower ones., the adjustment of the dosing strategymay provide partial protection of normal tissues.

DACA or other compound of the general formula I may be administered, forexample, in a divided dose over a period of up to 4 hours, for example 2to 4 hours, followed by a rest, for example for 3 to 4 weeks. The dosemay be divided into two to four administrations over the 2 to 4 hour orother administration period, and the first dose may be larger than theothers, that is, as a loading dose; administrations may be givenintravenously. For example, a short-term intravenous infusion of 10 to30 minutes (for example 15 minutes) may be used, followed by a furthersuch infusion after, for example, 1 hour. This schedule differs fromthat normally used for other cytotoxic agents, which involves periods ofintravenous infusion administered daily, for example for 3 to 7 days, orlong-term intravenous infusion over a number of days, for example for aweek.

We have found that DACA also has activity against melanoma cell linesand human tumour xenografts of these lines, and it is believed that thisactivity is improved by the same strategy.

The cytotoxicity of DACA was assessed in a panel of primary humanmelanoma cultures derived from fresh surgical melanoma specimens. IC₅₀values ranged from 0.2 o 1.5 μM, and a feature of the data was theability of DACA to kill much higher proportions of cells (>99%) in somecultures, as compared to a maximum of 90% for etoposide.

A further experiment was carried out using human melanoma line,implanted subcutaneously in nude (athymic) mice. Treatment was startedwhen the tumours were 4-7 mm in diameter. DACA was administered ip as adivided dose (2×100 mg/kg body weight at 0 and 60 min) and a secondsimilar administration (2×100 mg/kg) was given after 7 days. A growthdelay of 30 days was obtained.

The positive results achieved by DACA in these treatments is surprisingsince melanoma is more difficult to treat with chemotherapy than areother forms of tumour.

Moreover, Berger et al. (Berger D. P., Winterhalter B. R., Flebig H. H.,"Conventional chemotherapy" in "The Nude Mouse in Oncology Research",1st ed. London: CRC Press, 1991, 165-84, ed. Boven and Winograd) statesthat melanoma xenografts are resistant to treatment by doxorubicin andetoposide, so activity by a drug in this class is completely unexpected.A summary of the activity of various other agents against subcutaneousmelanoma xenografts growing in nude mice is given by Berger et al. asfollows:

    ______________________________________                                                          Percentage of xenografts                                    Drug              responding                                                  ______________________________________                                        Topo II inhibitors                                                            Doxorubicin        5% (total of 5 studies)                                    Etoposide          0% (2 studies)                                             Other drugs                                                                   Bleomycin          0%                                                         Cisplatin         13%                                                         Cyclophosphamide  11%                                                         Dacarbazine       17%                                                         5-Fluorouracil    14%                                                         Methotrexate       7%                                                         Mitomycin C       32%                                                         Vinblastine       10%                                                         Vincristine       43%                                                         ______________________________________                                    

In pharmacokinetic studies using radioactive (tritium-labelled) DACAhigh levels of active ingredient have been found in all tissues,including brain, with a long elimination t_(1/2) of 37-176 h. Asdetermined by HPLC, the tissue concentrations of DACA 1 h afterintraperitoneal administration of drug (400 μmol/kg) were 45, 185, 139,and 57 μmol/kg in brain, liver, kidney and heart, respectively. Thecorresponding AUC values (AUC=area under the plasma concentration-timecurve) were 218, 547, 492 and 147 μmol.h/l , respectively, as comparedto the plasma AUC of 26.6 μmol.h/l. DACA showed relatively high rates ofpassage across the blood brain barrier. We believe that administrationof DACA, especially at the new divided dose-high constituent dosefrequency regime mentioned above, will be helpful in combating braintumours. With the exception of nitrosoureas, few of the antitumouragents currently in use possess the physicochemical properties requiredfor adequate penetration of the blood-brain barrier (Greig M. H. (1987),Cancer Treat. Rep. 11: 157).

We also propose the use of DACA and related compounds with a "rescue"treatment with a second drug which by itself is not an active agent butwhich displaces DACA or the other compound from the DNA. ThisDNA-binder, or chemoprotector, should have a lower intrinsic toxicityand less efficient tissue distribution properties than the cytotoxicagent, thus sparing rapidly growing and highly vascularised normaltissues such as bone marrow from cytotoxic effects. Use of the newschedule of administration of DACA or other compound of the generalformula I or a physiologically tolerable acid addition salt or 1-N-oxidethereof, combined with "rescue" treatment with a chemoprotector, shouldespecially be mentioned. Timing of administration of the chemoprotectorwill depend on the pharmacokinetics of DACA or other drug used. Thechemoprotector may, for example, be administered at the same time as orup to 30 minutes after one or more of the constituent doses of a divideddose of that drug; by such means doses of, for example, 200 mg/kg oreven 300 mg/kg of DACA--doses which are normally toxic--may be possible.

The following Examples illustrate the invention.

EXAMPLES EXAMPLE 1

Activity of DACA against cultured multidrug resistant human leukaemiacells

Materials and Methods

Acridine carboxamide hydrochloride, synthesised in the Cancer ResearchLaboratory (Atwell G. J., et al., J. Med. Chem. 1987, 30, 664-669), andamsacrine isethionate, obtained from the Parke-Davis Division of theWarner-Lambert Company, Ann Arbor, USA, were dissolved in 50% v/vaqueous ethanol to make stock solutions of 2-5 mmol/l and stored at -20°C. Other cytotoxic drugs were available either from the NCI repository(Monks A., et al., J. Natl. Cancer Inst. 1991, 83, 757-766) or wereobtained as described in Marshall E. S., et al., J. Natl. Cancer Inst.1992, 84, 341-344 and Finlay, et al., Eur. J. Cancer Clin. Oncol. 1986,22, 655-662. Cell lines were from the NCI repository except for MM-96(Dr. R. Whitehead, Ludwig Institute, Melbourne, Australia), FME (Dr. K.M. Tveit, Norwegian Radium Hospital, Oslo, Norway) and Jurkat normal andmultidrug-resistant lines (Dr. K. Snow and Dr. W. Judd, Department ofCellular and Molecular Biology, University of Auckland). Melanoma tissuewas obtained from patients with pathologically confirmed metastatic andrecurrent melanomas under Auckland Hospital Ethical Committeeguidelines. Cells were released by digestion of tissue (at 50 mg.ml⁻¹)with collagenase (1 mg.ml⁻¹) and DNAase (50 μg.ml⁻¹) with continuousstirring at 37° C. for 1 to 2 hours, and cultured as previouslydescribed (Marshall E. S., et al., J. Natl. Cancer Inst. 1992, 84,341-344).

Tumour cell lines were cultured in 96-well plates. Growth of NCI celllines was assessed using sulphorhodamine B staining (Skehan P., et al.,J. Natl. Cancer Inst. 1990, 82, 1107-1112), that of the leukaemia lineswith (4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium (MTT) staining(Mosmann T., J. Immun. Methods 1983, 65, 55-63) and that of the primaryhuman tumour material by ³ H-thymidine incorporation (Marshall E. S., etal., J. Natl. Cancer Inst. 1992, 84, 341-344). Primary tumour materialwas cultured in 96-well plates in which the wells were coated withagarose to inhibit selectively the growth of normal cells. (Marshall E.S., et al., J. Natl. Cancer Inst. 1992, 84, 341-344). Primary cultureswere incubated at 37° C. in sealed perspex boxes containing a humidifiedatmosphere of 5% CO₂ and 5% O₂ in nitrogen for 7 days. 5-Methyl- ³H!-thymidine (20 Ci.mmol⁻¹ ; 0.04 μCi per well), thymidine and5-fluorodeoxyuridine (each at final concentrations of 0.5 μM) were addedin medium to cultures (20 μl per well) 24 h before terminating thecultures. Cells were aspirated on to glass fibre filters using amultiple automated sample harvester (LKB Wallac OY Beta Harvester). Thefilter discs were washed for 15 seconds with water, dried, and theamount of tritium retained quantified by liquid scintillation.

IC₅₀ values were defined as in Paull K. D., et al., J. Natl. CancerInst. 1989, 81:1088-1092, where growth, as indicated by staining orthymidine incorporation, corresponded to 50% of that of the controlcultures. DELTA values were determined for groups of logarithmic IC₅₀values as deviations from the mean, with positive DELTA valuesrepresenting higher drug sensitivity relative to the mean. Variances ofDELTA values were expressed as standard deviations in log₁₀ units.Comparison of DELTA values was made using Pearson correlationcoefficients. Resistance factors were defined as the ratios of IC₅₀values between the resistant line and the parent line. Statisticalevaluation was performed either with NCI programmes, with RS/1 software(BBN Research Systems, Cambridge, Mass., USA), or with Sigmaplot (JandelScientific, San Rafael, Calif., USA).

Results

The effect of DACA on the growth of cultured cells was assessed bycontinuous drug exposure. DACA inhibited the growth of two human JurkatT cell leukaemia lines, one diploid (L) and the other tetraploid (B1),with IC₅₀ values each of 380 nM. These values were similar to those ofother human leukaemia cell lines, which ranged from 290 to 760 nM(CEM-CCRF, 410 nM; MOLT-4, 290 nM; Daudi, 400 nM; Raji, 370 nM, U937,590 nM; HL-60, 620 nM; K-562, 760 nM). Four multidrug-resistant celllines, developed from JL and JB1 by in vitro exposure to increasingconcentrations of doxorubicin (L_(D) and B1_(D)) or amsacrine (L_(A) andB1_(A)) (Finlay G. J., et al., J. Natl. Cancer Inst. 1990, 82, 662-667,Snow K., et al., Br. J. Cancer 1991, 63, 17-28) were tested.

DACA was compared with six other drugs including four topo II poisons,doxorubicin, mitozantrone, etoposide and amsacrine. Resistance factorsfor the topo II poisons were consistently higher than those for DACA(Table 1). In contrast, the topoisomerase I poison camptothecin showedno cross resistance, and the mitotic inhibitor vincristine showed adifferent pattern of resistance with the B1_(D) line having the highestresistance (Table 1).

                  TABLE 1                                                         ______________________________________                                        Drug-resistant Jurkat Leukaemia sublines.                                     Resistance factors                                                            doxo-    mitoxan- etopo-  amsa-      campto-                                                                             vincris-                           rubicin  trone    side    crine                                                                              DACA  thecin                                                                              tine                               ______________________________________                                        L.sub.A                                                                            3.8     42       11    130  2.0   1.0   1.5                              L.sub.D                                                                            16      160      93    110  2.5   0.97  3.6                              B1.sub.A                                                                           11      59       22    240  3.9   0.48  2.0                              B1.sub.D                                                                           15      8.4      83    8.8  1.9   0.86  10                               ______________________________________                                    

One method of providing a visual comparison of the patterns ofresistance is to plot DELTA values (Paull K. D., et al., J. Natl. CancerInst. 1989, 81, 1088-1092) where the differences in bar lengths are usedas a measure of relative resistance. FIG. 1 shows a comparison of DELTAvalues in log mean graph format for DACA, amsacrine, etoposide,doxorubicin, vincristine, 5-fluorouracil, camptothecin and mitozantronefor the panel of multidrug resistant Jurkat leukaemia lines using MTTstaining; JL/AMSA and JB/AMSA were selected for resistance to amsacrineand JL/DOX and JB/DOX were resistant to doxorubicin. DACA clearly showsa pattern distinct from doxorubicin.

A second method of comparing agents is to plot resistance factors forone of the lines against another. Since the Jurkat lines exhibitedpredominantly "altered topoisomerase" resistance (Finlay G. J., et al.,J. Natl. Cancer Inst. 1990, 82, 662-667, Sugimoto Y., et al., CancerRes. 1990, 50, 7962-7965), the resistance factors for one of these(L_(A)) was plotted versus the resistance factors for a P-glycoproteinpositive multidrug resistant P388 Leukaemia line (P/DACT) which exhibitstransport resistance (Baguley B. C., J. Natl. Cancer Inst. 1990, 82,398-402). The results (FIG. 2) indicate that DACA is unique whencompared to other topo II agents in that it is able to overcome twodifferent multidrug resistance mechanisms. Qualitatively similar graphsare obtained when the resistance factors of the other resistant Jurkatlines are plotted on the abscissa, or those from a P-glycoproteinpositive, vinblastine resistant human leukaemia line (CEM/VLB₁₀₀) (QianX., Beck W. T., Cancer Res. 1990, 50, 1132-1137) are plotted on theordinate.

DACA was also compared with three other topo II agents using a panel ofcell lines (data provided by Dr. Ken Paull from the National CancerInstitute, USA) encompassing a number of tumour types, and using proteinstaining. The mean IC₅₀ for DACA was 2,100 nM, as compared withamsacrine (520 nM), etoposide (21,000 nM) and doxorubicin (140 nM). Theresults, presented as DELTA plots, are compared with corresponding plotsfor three other topoisomerase II poisons in FIG. 3. The variance ofDELTA values was considerably smaller for DACA (0.24 units) than it wasfor amsacrine (0.61 units) etoposide (0.55 units) or doxorubicin (0.44units). The differences in DELTA values for amsacrine, etoposide anddoxorubicin for primary human cultures imply that intrinsic resistancemechanisms exist and are partially overcome with DACA.

DACA was also compared in a series of 12 primary melanoma cultures.Tissue was excised from human malignant melanomas and cultured using amodified 96-well assay system in which the cells were cultured onagarose and assayed for proliferation using the ³ H-thymidineincorporation assay as described in Marshall E. S., et al., J. Natl.Cancer Inst. 1992, 84, 341-344. The mean IC₅₀ for DACA was 590 nM, ascompared with amsacrine (128 nM), etoposide (2,200 nM) and doxorubicin(56 nM). DELTA values for DACA, amsacrine, etoposide and doxorubicin arecompared in FIG. 4. The variance of DELTA values was smaller for DACA(0.39 units) than for amsacrine (0.54 units), etoposide (0.66 units) ordoxorubicin (0.63 units). The differences in DELTA values for amsacrine,etoposide and doxorubicin for primary human cultures again imply thatintrinsic resistance mechanisms exist and are partially overcome withDACA.

EXAMPLE 2

Activity of DACA against advanced colon 38 and melanoma in mice

Materials and Methods

Colon 38 carcinoma was obtained from the Mason Research Institute(Worchester, Mass., USA) and was grown in BDF₁ hosts. Tumour fragments(1 mm³) were implanted subcutaneously in anaesthetised mice. Tumours hadgrown to the appropriate size 9 days after implantation. A melanomatumour line (WADH) was developed in the Cancer Research Laboratory.Tumour cells were grown in culture and 1×10⁶ cells were implantedintradermally into the flank of nude (C57BI/J genetic background) mice.Mice were grown under sterile surroundings until tumours were ofappropriate size.

Tumours were measured 3× (colon 38) or 2× (xeno-graft) weekly withcallipers and tumour volumes calculated as 0.52a² b, where a and b werethe minor and major tumour axes. Tumour growth delays were measured at atime when tumour volumes of treated and control animals had increased by4-fold.

Results

The effect of DACA on the growth of advanced colon 38 tumours in micewas investigated by implanting tumour fragments subcutaneously andallowing them to grow until they had reached a diameter of 5-8 mm. I.p.treatment of mice with a single maximum tolerated dose of DACA (150mg/kg body weight), a treatment which was known to induce cures ofintravenously implanted Lewis lung tumours (Finlay G. J., Baguley B. C.,Eur. J. Cancer Clin. Oncol. 1989, 25, 271-277) caused only a slightgrowth delay (5 days; FIG. 5). However, when a divided dose (200 mg/kg)was administered over a period of 0.5-4 hours, greater delays wereunexpectedly observed (Table 2). Repetition of these divided dosesprovided a substantial growth delay (23 days; FIG. 5) which was longerthan that obtained with the maximum tolerated dose of amsacrine (2days), cyclophosphamide (6.5 days) or 5-fluorouracil (13 days).

                  TABLE 2                                                         ______________________________________                                        Tumour growth delays (colon 38) treated with DACA                             Total dose                                                                            Schedule           Growth delay (days)                                ______________________________________                                        100   ip    single dose (SD)   4                                              150   ip    SD                 5                                              150 × 3                                                                       ip    SD every week × 3                                                                          7                                              150   ip    2 doses, 0, 60 min 5                                              200   ip    SD                 toxic                                          200   ip    2 doses, 0, 30 min 7                                              200   ip    2 doses, 0, 60 min 10, 12 (2 expts)                               200   ip    2 doses, 0, 24 hours                                                                             6                                              200   ip    4 doses, 0, 30, 60, 90 min                                                                       6                                              200   iv    1 hour infusion    7                                              200   iv    3 hour infusion    6.5                                            200 × 3                                                                       ip    (4 doses, 0, 30, 60, 90 min) × 3                                                           23                                             ______________________________________                                         Note: the 4 dose schedule was 65 + 45 + 45 + 45 mg/kg                    

A further experiment was carried out using human melanoma line,implanted subcutaneously in nude (athymic) mice using an inoculation ofone million cells of a human melanoma cell line designated WADH.Treatment was started when the tumours were 4-7 mm in diameter. DACA wasadministered ip as a divided dose (2×100 mg/kg body weight at 0 and 60min) and a second similar administration (2×100 mg/kg) was given after 7days. A growth delay of 30 days was obtained (FIG. 6).

EXAMPLE 3

Exploitation of the self-inhibitory properties of a drug in the therapyof solid tumours

One of the characteristics of solid tumours is that because of the poorvascularisation, oxygen, nutrients and chemotherapeutic drugs mustdiffuse for longer distances than they do in normal tissue (Wilson W.R., Denny W. A., Radiation Research: a Twentieth Century Perspective,1st ed. v. 2. New York: Academic Press, 1992:796-801). In the case ofantitumour agents, a gradient of drug concentration is established withthe lowest drug concentration at greatest distances from the capillary.Since in all cases examined so far with existing clinical agents,cytotoxicity is related in a positive fashion to drug concentration, itfollows that those areas most remote from the tumour blood supply areprotected from drug cytotoxicity, a so-called "pharmacologicalsanctuary".

DACA is a DNA intercalating agent which acts on topo II and has theunusual property of inhibiting its own toxicity at concentrations above5 μm. It also inhibits the formation of DNA-protein cross-links above 5μM, consistent with the hypothesis that self-inhibition of DNA-proteincross-links is related to self-inhibition of toxicity. A simple modelfor this behaviour is that in order for topo II to form its complex withDNA (i.e. to form DNA-protein cross-links) it requires the presence of aDNA-drug complex (probability=p), surrounded on each side by drug-freeDNA (probability=(1-p)). It follows that the probability of forming aproductive complex is p(1-p)². When this function is plotted againstexperimental cytotoxicity data for DACA (Haldane A., et al., CancerChemother. Pharmacol. 1992, 29, 475-479), a good approximation isobtained (FIG. 7).

FIG. 7 can also be plotted as toxicity versus cell-associated drug(using unpublished data from the Cancer Research Laboratory whichrelates external drug concentration to cell-associated drug). It can beseen from FIG. 7 that if a tumour concentration gradient is establishedwhereby the area of the tumour closest to the capillary has, forexample, a concentration of 1800 μmol/kg, areas of the tumour which aremore remote from the capillary, although having a lower drugconcentration, will have higher cytotoxicity. Furthermore, host tissues,which have good blood supplies, will have high tissue drugconcentrations and thus lower cytotoxicity. By this principle, DACA (andother compounds of this general class) could have a selectivitymechanism for solid tumours which is not possessed by other agents.

The practical application of this hypothetical situation requires thatfree drug plasma concentrations (and corresponding tissue concentrationsof drug) fall into the range which will provide selectivity (i.e.greater than 1000 μmol/kg tissue). Preliminary results (Dr. JamesPaxton, personal communication) indicate that when DACA is administeredat a maximally tolerated single drug dose (150 mg/kg body weight), drugconcentrations in normal tissues (e.g. liver, spleen) slightly exceed1000 μmol/kg. This principle may be exploited further by drug design orby combining DACA administration with that of a second chemoprotectoragent which increases the self-inhibition of DACA (i.e. the descendingpart of the curve in FIG. 7) and thus lowers the average tissue drugconcentration required for the application of this principle.

We claim:
 1. A method for the treatment of advanced colon cancer, whichmethod comprises administering to a patient in need thereof, by adivided dose schedule, a therapeutically effective amount of a compoundwhich is an acridine carboxamide of formula I': ##STR5## wherein R₁ isselected from the group consisting of H, CH₃ and NHR₀, wherein R0 isselected from the group consisting of H, COCH₃, SO₂ CH₃, COPh, SO₂ Phand C₁ -C₄ alkyl which is unsubstituted or bears a substituent selectedfrom the group consisting of hydroxy, C₁ -C₄ alkoxy and amino;n is aninteger from 2 to 6; R₈ is H or is one or two substituents selected fromthe group consisting of CH₃, OCH₃, halogen, CF₃, NO₂, NH₂, NHCOCH₃ andNHCOOCH₃ at positions 1-3 and 5-8; and Y is selected from the groupconsisting of C(NH)NH₂, NHC(NH)NH₂ and NR₄ R₅ wherein each of R₄ and R₅is H or C₁ -C₄ alkyl unsubstituted or substituted by hydroxy or amino;ora physiologically tolerable acid addition salt or N-oxide thereof; thedivided dose schedule comprising a first administration and a secondadministration of the said compound wherein the second administrationcommences 15 minutes or more, but less than one day, after commencementof the first administration.
 2. A method according to claim 1 wherein,in Formula I':R₁ is H or NH₂ n is 2 R₅ is one or two substituentsselected from the group consisting of 1-NO₂, 5-NO₂, 6-NO₂, 7-NO₂, 8-NO₂,5-CH₃, 6-CH₃ and 5-Cl; and Y is selected from the group consisting ofNHC(NH)NH₂, N(CH₃)₂ and NHCH₂ CH₂ OH.
 3. A method according to claim 1wherein the acridine carboxamide of formula (I') is N-2-(dimethylamino)ethyl!acridine-4-carboxamide.
 4. A method according toclaim 1 wherein the compound is the dihydrochloride salt of N-2-(dimethylamino)ethyl!acridine-4-carboxamide.
 5. A method according toclaim 1 which comprises administering, simultaneously or sequentiallywith the said compound, a second component selected from the groupconsisting of cisplatin, cyclophosphamide, bleomycin, carboplatin,5-fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, vincristine,vinblastine and vindesine.
 6. A method according to claim 1 wherein thedivided dose schedule comprises at least two administrations of drugover a period of up to 4 hours.
 7. A pharmaceutical composition for usein the treatment of advanced colon cancer, comprising(i) a firstcomponent which is an acridine carboxamide of formula (I'): ##STR6##wherein R₁ is selected from the group consisting of H, CH₃ and NHR₀,wherein R₀ is selected from the group consisting of H, COCH₃, SO₂ CH₃,COPh, SO₂ Ph and C₁ -C₄ alkyl which is unsubstituted or bears asubstituent selected from the group consisting of hydroxy, C₁ -C₄ alkoxyand amino; n is an integer from 2 to 6; R₈ is H or is one or twosubstituents selected from the group consisting of CH₃, OCH₃, halogen,CF₃, NO₂, NH₂, NHCOCH₃ and NHCOOCH₃ at positions 1-3 and 5-8; and Y isselected from the group consisting of C(NH)NH₂, NHC(NH)NH₂ and NR₄ R₅wherein each of R₄ and R₅ is H or C₁ -C₄ alkyl unsubstituted orsubstituted by hydroxy or amino; or a physiologically tolerable acidaddition salt or N-oxide thereof;and (ii) a second component which isselected from the group consisting of cisplatin, cyclophosphamide,bleomycin, carboplatin, 5-fluorouracil, 5-fluorodeoxyuridine,methotrexate, taxol, vincristine, vinblastine and vindesine; the saidfirst and second components being present together in the sameformulation.
 8. A composition according to claim 7 wherein, in formula(I'),R₁ is H or NH₂ n is 2 R₈ is one or two substituents selected fromthe group consisting of 1-NO₂, 5-NO₂, 6-NO₂, 7-NO₂, 8-NO₂, 5-CH₃, 6-CH₃and 5-Cl; and Y is selected from the group consisting of NHC(NH)NH₂,N)CH₃)₂ and NHCH₂ CH₂ OH.
 9. A composition according to claim 7 whereinthe derivative of formula (I') is N-2-(dimethylamino)ethyl!acridine-4-carboxamide.
 10. A compositionaccording to claim 7 wherein the compound is the dihydrochloride salt ofN- 2-(dimethylamino)ethyl!acridine-4-carboxamide.
 11. A method accordingto claim 1 wherein the divided dose schedule comprises a thirdadministration and optional further administrations of the saidcompound, the said third and optional further administrations eachcommencing 15 minutes of more, but less than one day, after commencementof the previous administration.
 12. A method according to claim 1wherein the divided dose schedule comprises at least 2 administrationsof the said compound over a period of from 30 minutes to 4 hours.
 13. Amethod according to claim 1 wherein the divided dose schedule comprises2 to 4 administrations of the said compound over a period of 2 to 4hours.